Our long-term objective is to define in molecular terms the cellular proteins that form, direct and regulate membrane traffic in eukaryotic cells. Our focus is on clathrin vesicles, a key transporter of receptors in eukaryotic cells. Clathrin coated vesicles play important cellular roles in the internalization of nutrients and signals from the extracellular milieu. Our experimental strategy is to use Dictyostelium as a model system. Our two specific aims are designed to answer the important questions of how and where clathrin lattices assemble in living cells. 1. We will determine the contribution of the clathrin light chain (CLC) to coated vesicle structure and function in living cells. (A) We will assess the in vitro assembly properties of CLC-deficient triskelions purified from CLC null cells. By determining the clathrin lattice structure and co-localization with Assembly Proteins in CLC null cells, the in vivo assembly properties of CLC-deficient triskelions on the plasma membrane will be defined. (B) Three independent approaches utilizing protein purification, two-hybrid analysis, and a high-copy genetic suppressor screen, will be used to identify proteins and genes that bind to or interact genetically with the clathrin light chain. The function and contribution of these binding and genetic partners to clathrin function will be determined. 2. We will determine the mechanism of regulation of clathrin lattice formation by the assembly protein CALM. CALM, clathrin assembly myeloid leukemia gene was identified as a gene whose disruption is associated with the disease myeloid leukemia. Possible roles for CALM as an obligate initiator of clathrin assembly on the plasma membrane or as a modulator of clathrin assembly will be distinguished. (A) Using phenotypic analysis of CALM-minus Dictyostelium, we will assess the contribution of CALM to trafficking of receptors that utilize clathrin-mediated pathways. Double mutant analysis of cells will test the possibility that CALM collaborates with proteins of similar domain structure to accomplish its intracellular role. (B) The requirement for CALM for the proper formation of clathrin assembly and vesicle formation will be assessed in clathrin-minus mutants. The order of CALM and clathrin assembly on the plasma membrane will be established. (C) Defined fragments of the CALM protein will be assessed for PIP2 and clathrin-binding. Expression of these domains in CALM minus cells will define the contribution of these domains to the proper localization and in vivo function of the CALM protein.